曲古抑菌素A对大鼠气管干细胞增殖分化的影响

发布时间：2017-12-31 05:33

本文关键词：曲古抑菌素A对大鼠气管干细胞增殖分化的影响　出处：《中国医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的目前,在气管干细胞研究中,我们建立了氟脲嘧啶(Fluorouracil,5-Fu)引发离体气管损伤修复模型,论证了气管干细胞位于具有5-FU抗性的G0期细胞中。为阐明气管干细胞增殖分化的调控机制,我们已检测了wnt信号中的wnt1,β-catenin,cyclinD1等成员在气管干细胞增殖分化过程中的时空变化情况,证明了Wnt/β-catenin信号传导途径参与调控气管干细胞增殖分化全过程。另外,我们还观察到了HDAC1在气管干细胞增殖分化过程中的动态表达情况。本文结合前段我研究组工作基础,利用TSA抑制HDAC1的作用,采用形态学、免疫组化及western blotting方珐观察其在5-Fu模型中对气管干细胞增殖分化的影响。方法 1、制备气管损伤模型及剥离上皮取约200克左右的Wistar大鼠,雌雄不限(中国医科大学实验动物中心提供),腹腔注射10%水合氯醛0.4ml/100g,无菌条件下取出气管,无菌PBS反复冲洗,洗净血液和粘液,置于DMEM/F12培养液中(含10%胎牛血清),于培养液中加入终浓度为100mg/ml的5-Fu,37℃,5%CO2孵育12小时,弃去上述培养液,换成新鲜DMEM/F12液(含10%胎牛血清),同时加入终浓度为200nM的TSA继续培养,于换液后3、6、12、24、48小时分别10%中性福尔马林固定,石蜡包埋,制成4μm厚的组织切片。同时各时间点取气管组织,放于Eppendorf管中,-70℃保存。 2、HE染色动态观察各时间点气管粘膜上皮组织学形态改变。 3、利用免疫组织化学染色观察没有TSA作用时HDAC1在5-Fu模型中的表达。 4、Western blotting检测TSA对各时间点HDAC1的表达量变化的影响。结果 1、HE:在没有TSA作用情况下,去除5-Fu作用后0h,大部分气管上皮脱落,残留间隔分布的裸核样细胞(即G0期细胞);去除5—FU后恢复3h,裸核细胞消失,细胞呈扁平状,细胞核变长,细胞浆伸展几乎可以将基底膜覆盖;6h,细胞核变圆,有处可见变为立方上皮,细胞数增多;12小时,上皮完全变为立方上皮,胞核增大,胞浆增多,上皮细胞数目继续增多;恢复至24小时,细胞表面出现多量纤毛;恢复48h,气管上皮接近恢复假复层结构。在有TSA作用情况下,去除5-Fu作用后0-12小时,基底膜上残留裸核细胞基本没有变化,但是数量稍有增加;24-48小时,裸核细胞消失,细胞成扁平状,细胞核变长,细胞浆伸展覆盖基底膜。 2、免疫组化检测结果:HDAC1呈胞核阳性。无TSA作用时经5-Fu作用后0小时,未见HDAC1阳性细胞;去除5-Fu后3—6小时,仍未见明显HDAC1阳性细胞;12小时,HDAC1阳性细胞数明显增多,并可见几个HDAC1阳性细胞围绕着底部一个HDAC1阴性细胞和多个HDAC1阳性细胞间夹着几个HDAC1阴性细胞分布的现象;24小时,HDAC1阳性细胞数最多,累及全层。48小时,HDAC1阳性细胞数略减少,阳性细胞分布集中于假复层结构顶部即靠近管腔侧。TSA作用后,基本没有HDAC1阳性细胞出现。 3、Western blotting检测结果:无TSA作用时,HDAC1正常无表达,去除5—FU后0小时无表达,3、6、24小时表达量依次递增,24小时达高峰,48小时表达量略减少。TSA作用时,HDAC1表达明显受到抑制。结论 1、TSA可以阻断经过5-Fu处理后的气管上皮损伤修复过程,抑制气管干细胞的分化,造成气管干细胞的增殖堆积。 2、本研究有可能为解决干细胞供体不足,提供一种新的方法,可能将对干细胞的来源和实际应用带来巨大优势。
[Abstract]:objective
At present, the tracheal stem cell research, we established the fluorouracil (Fluorouracil, 5-Fu) induced tracheal regeneration model, demonstrated that tracheal stem cells located with 5-FU resistant G0 cells. To elucidate the regulatory mechanism of tracheal stem cell proliferation and differentiation, we have detected the Wnt signal in Wnt1. Beta -catenin, cyclinD1 and other members of the tracheal stem cell proliferation and differentiation process of temporal and spatial change in the situation that Wnt/ beta -catenin signaling pathway involved in the regulation of cell proliferation and differentiation process of tracheal stem. In addition, we also observed the expression of HDAC1 in tracheal stem cell proliferation and differentiation in the dynamic process. This paper combined with the preceding research group I the work of the foundation, the inhibition of HDAC1 by TSA, the morphological, immunohistochemical and Western blotting methods to observe the effect of stem cells on proliferation and differentiation of trachea in 5-Fu model.
Method
1, the preparation of the model of tracheal injury and the exfoliation epithelium
Take about 200 grams of Wistar rats, male and female (provided by the experimental animal center of China Medical University), intraperitoneal injection of 10% chloral hydrate 0.4ml/100g, trachea removed under sterile conditions, aseptic PBS repeated washing, clean the blood and mucus in DMEM/F12 medium (containing 10% FBS), in the culture medium with a final the concentration of 100mg/ml 5-Fu, 37 C, 5%CO2 incubated for 12 hours, discard the culture liquid, replaced with fresh DMEM/F12 solution (containing 10% FBS), while continuing to cultivate 200nM TSA with the final concentration, to change liquid after 3,6,12,24,48 hours respectively in 10% neutral formalin fixed, paraffin embedded, made of 4 m thick tissue sections. At the same time each time point for tracheal tissue, put in the Eppendorf tube, -70 C preservation.
2, HE staining was used to observe the histological changes of tracheal epithelium at every time point.
3, immunohistochemical staining was used to observe the expression of HDAC1 in the 5-Fu model without the effect of TSA.
4, Western blotting detected the effect of TSA on the changes in the expression of HDAC1 at each time point.
Result
1, HE: without the effect of TSA, the removal of 5-Fu after 0h, most of the tracheal epithelial cells, residual naked nucleus intervals (G0 cells); removal of 5 - FU recovery after 3h, bare cells disappeared, the cells were flat, the nucleus becomes long, cytoplasm stretching almost the basement membrane covering; 6H, the nucleus became round, are visible into cuboidal epithelium, the number of cells increased; 12 hours, completely turned into cuboidal epithelium, enlarged nuclei, cytoplasm, epithelial cell number continues to increase; recovery to 24 hours, many ciliated cell surface; 48h recovery, tracheal epithelium to restore the pseudostratified structure. In the case of TSA, 0-12 hours after the removal of 5-Fu, the basement membrane residual bare cells did not change, but the number increased slightly; 24-48 hours, bare cells disappeared, flattened cells, the nucleus becomes long, cytoplasm extends over basal membrane.
2, the results of immunohistochemistry: the HDAC1 was located in the nucleus. TSA positive effect after 0 hours after 5-Fu, no HDAC1 positive cells; the removal of 3 - 6 hours after 5-Fu, still no obvious HDAC1 positive cells; 12 hours, the number of HDAC1 positive cells increased obviously, and visible a few HDAC1 positive cells around the bottom a negative HDAC1 cells and a number of HDAC1 positive cells between several negative cells distribution HDAC1; 24 hours, the number of HDAC1 positive cells, full-thickness.48 hours, the number of HDAC1 positive cells reduced slightly, the positive cells were concentrated on pseudostratified structure that is close to the top of the lumen side after.TSA, basic no HDAC1 positive cells appeared.
3, Western blotting test results: when there was no TSA action, HDAC1 was normal without expression. After 5 to FU removal, it didn't express at 0 hours, 3,6,24 hours increased gradually, reached peak at 24 hours, and decreased at 48 hours, HDAC1 expression was significantly inhibited when.TSA was used.
conclusion
1, TSA can block the repair process of tracheal epithelium injury after 5-Fu treatment, inhibit the differentiation of tracheal stem cells and cause the proliferation and accumulation of tracheal stem cells.
2, this study may provide a new way to solve the deficiency of stem cell donor, which may bring a great advantage to the source and practical application of stem cells.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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